2CS-CHXT Operon Signature of Chlorhexidine Tolerance among Enterococcus faecium Isolates

Abstract

Chlorhexidine (CHX) is a broad-spectrum antiseptic widely used in community and clinical contexts for many years that has recently acquired higher relevance in nosocomial infection control worldwide. Despite this, CHX tolerance among Enterococcus faecium bacteria, representing one of the leading agents causing nosocomial infections, has been poorly understood. This study provides new phenotypic and molecular data for better identification of CHX-tolerant E. faecium subpopulations in community and clinical contexts. The chlorhexidine MIC (MIC_CHX) distribution of 106 E. faecium isolates suggested the occurrence of tolerant subpopulations in diverse sources (human, animal, food, environment) and phylogenomic backgrounds (clades A1/A2/B), with predominance in clade A1. They carried a specific variant of the 2CS-CHX^T operon, identified here. It encodes glucose and amino acid-polyamine-organocation family transporters, besides the DNA-binding response regulator ChtR, with a P102H mutation previously described only in CHX-tolerant clade A1 E. faecium, and the ChtS sensor. 2CS-CHX^T seems to be associated with three regulons modulating diverse bacterial biological functions. Combined data from normal MIC distribution and 2CS-CHX^T operon characterization support a tentative epidemiological cutoff (ECOFF) of 8 mg/liter to CHX, which is useful to detect tolerant E. faecium populations in future surveillance studies. The spread of tolerant E. faecium in diverse epidemiological backgrounds calls for the prudent use of CHX in multiple contexts.IMPORTANCE Chlorhexidine is one of the substances included in the World Health Organization's list of essential medicines, which comprises the safest and most effective medicines needed in global health systems. Although it has been widely applied as a disinfectant and antiseptic in health care (skin, hands, mouthwashes, eye drops) since the 1950s, its use in hospitals to prevent nosocomial infections has increased worldwide in recent years. Here, we provide a comprehensive study on chlorhexidine tolerance among strains of Enterococcus faecium, one of the leading nosocomial agents worldwide, and identify a novel 2CS-CHX^T operon as a signature of tolerant strains occurring in diverse phylogenomic groups. Our data allowed for the proposal of a tentative epidemiological cutoff of 8 mg/liter, which is useful to detect tolerant E. faecium populations in surveillance studies in community and clinical contexts. The prediction of 2CS-CHX^T regulons will also facilitate the design of future experimental studies to better uncover chlorhexidine tolerance among E. faecium bacteria.

Keywords: 2CS-CHXT operon; Enterococcus faecium; chlorhexidine tolerance; epidemiological cutoff.

FIG 1

Distribution of the Enterococcus faecium studied by different chlorhexidine MICs. The graph fitted curves and limits of 95% of wild-type population (L95-WT) were drawn based on the calculations of the ECOFFinder tool. (I) Distribution of the 106 E. faecium isolates studied (all clades). (II) Distribution of clade A1 E. faecium isolates (n = 48; L95-WT ≤ 16 mg/liter). (IIIa) Distribution of raw data for clade A2 E. faecium isolates (n = 43). (IIIb) Distribution of clade A2 E. faecium isolates (n = 27) without isolates presenting MIC values corresponding to the second mode and higher. The removal of such isolates is necessary for the ECOFFinder tool be able to propose the L95-WT, in this case ≤8 mg/liter. (IV) Distribution of clade B E. faecium isolates (n = 15; L95-WT ≤ 16 mg/liter). The DIST.NORM Excel 16.19 function indicates that in all cases the probability of occurrence of an isolate with an MIC of >16 mg/liter is 0% and variable among clades in the range of MIC > 8 and ≤ 16 mg/liter as follows: 99.9% for clade A1, 0% for clade A2, and 0.00014% for clade B. MIC = 2 mg/liter represents MIC ≤ 2 mg/liter.